Ligand-specific dose-response of heterologously expressed olfactory receptors.

Primary olfactory neuronal cultures exposed to odorant stimulation have previously exhibited concentration-related effects in terms of intracellular cAMP levels and adenylate cyclase activity [Ronnett, G.V., Parfitt, D.J., Hester, L.D. & Snyder, S.H. (1991) PNAS88, 2366-2369]. Maximal stimulation occurred for intermediate concentrations, whereas AC activity declined for both low and high odorant concentrations. We suspected that this behavior might be ascribed to the intrinsic response of the first molecular species concerned by odorant detection, i.e. the olfactory receptor itself. In order to check this hypothesis, we developed an heterologous expression system in mammalian cells to characterize the functional response of receptors to odorants. Two mammalian olfactory receptors were used to initiate the study, the rat I7 olfactory receptor and the human OR17-40 olfactory receptor. The cellular response of transfected cells to an odorant stimulation was tested by a spectrofluorimetric intracellular calcium assay, and proved in all cases to be dose-dependent for the known ligands of these receptors, with an optimal response for intermediate concentrations. Further experiments were carried out with the rat I7 olfactory receptor, for which the sensitivity to an odorant, indicated by the concentration yielding the optimal calcium response, depended on the carbon chain length of the aldehydic odorant. The response is thus both ligand-specific and dose-dependent. We thus demonstrate that a differential dose-response originates from the olfactory receptor itself, which is thus capable of efficient discrimination between closely related agonists.     

Odorant response assays for a heterologously expressed olfactory receptor

Odorant responsiveness of a mouse olfactory receptor, mOR-EG, was investigated in various heterologous cells using a variety of detection methods. Odorant-induced Ca(2+) response was observed in HEK293 cells that coexpressed mOR-EG and the promiscuous G protein, G alpha 15. Without G alpha 15, a robust increase in cAMP level was observed upon odorant-stimulation in various mammalian cells. A luciferase reporter gene assay using zif268 promoter was adopted to amplify the cAMP signals. In Xenopus laevis oocytes, odorant-stimulated currents were recorded when mOR-EG cRNA was co-injected with either G alpha 15 or cAMP-dependent channel. These results suggest that odorant responsiveness can be monitored via a signaling pathway mediated by endogenous G alphas or transfected G alpha 15 in heterologous cell systems. Various functional assays for a heterologously expressed olfactory receptor reported in this study, are potentially useful for high-throughput ligand screening and functional analyses of hundreds of olfactory receptors.     

Comparison of heterologously expressed human cardiac and skeletal muscle sodium channels.

In this study we have expressed and characterized recombinant cardiac and skeletal muscle sodium channel alpha subunits in tsA-201 cells under identical experimental conditions. Unlike the Xenopus oocyte expression system, in tsA-201 cells (transformed human embryonic kidney) both channels seem to gate rapidly, as in native tissue. In general, hSkM1 gating seemed faster than hH1 both in terms of rate of inactivation and rate of recovery from inactivation as well as time to peak current. The midpoint of the steady-state inactivation curve was approximately 25 mV more negative for hH1 compared with hSkM1. In both isoforms, the steady-state channel availability relationships ("inactivation curves") shifted toward more negative membrane potentials with time. The cardiac isoform showed a minimal shift in the activation curve as a function of time after whole-cell dialysis, whereas hSkM1 showed a continued and marked negative shift in the activation voltage dependence of channel gating. This observation suggests that the mechanism underlying the shift in inactivation voltage dependence may be similar to the one that is causing the shift in the activation voltage dependence in hSkM1 but that this is uncoupled in the cardiac isoform. These results demonstrate the utility and limitations of measuring cardiac and skeletal muscle recombinant Na+ channels in tsA-201 cells. This baseline characterization will be useful for future investigations on channel mutants and pharmacology.     

Chemical and steady-state kinetic analyses of a heterologously expressed heme dependent chlorite dismutase

Chlorite dismutase carries out the heme-catalyzed decomposition of ClO2- to Cl- and O2, an unusual transformation with biotechnological and bioremediative applications. The enzyme has been successfully overexpressed for the first time in highly functional form in Escherichia coli and its steady state kinetics studied. The purified enzyme is abundant (55 mg/L cell culture), highly active (approximately 4.7 x 10(3) micromol of ClO2- min(-1) mg(-1) subunit) and nearly stoichiometric in heme; further, it shares spectroscopic and physicochemical features with chlorite dismutases previously isolated from three organisms. A careful study of the enzyme's steady state kinetics has been carried out. ClO2- consumption and O2 release rates were measured, yielding comparable values of kcat (4.5 x 10(5) min(-1)), K(m) (approximately 215 microM), and kcat/Km (3.5 x 10(7) M(-1) s(-1) via either method (4 degrees C, pH 6.8; all values referenced per heme-containing subunit). ClO2-:O2 stoichiometry exhibited a 1:1 relationship under all conditions measured. Though the value of kcat/Km indicates near diffusion control of the reaction, viscosogens had no effect on k(cat)/K(m) or V(max). The product O2 did not inhibit the reaction at saturating [O2], but Cl- is a mixed inhibitor with relatively high values of KI (225 mM for enzyme and 95.6 mM for the enzyme-substrate complex), indicating a relatively low affinity of the heme iron for halogen ions. Chlorite irreversibly inactivates the enzyme after approximately 1.7 x 10(4) turnovers (per heme) and with a half-life of 0.39 min, resulting in bleaching of the heme chromophore. The inactivation K(I) (K(inact)) of 166 microM is similar in magnitude to Km, consistent with a common Michaelis complex on the pathway to both reaction and inactivation. The one-electron peroxidase substrate guaiacol offers incomplete protection of the enzyme from inactivation. Mechanisms in keeping with the available data and the properties of other well-described heme enzymes are proposed.     

Purification and characterization of heterologously expressed nitrilases from filamentous fungi

Nitrilases from Aspergillus niger CBS 513.88, A. niger K10, Gibberella moniliformis, Neurospora crassa OR74A, and Penicillium marneffei ATCC 18224 were expressed in Escherichia coli BL21-Gold (DE3) after IPTG induction. N. crassa nitrilase exhibited the highest yield of 69,000 U L(-1) culture. Co-expression of chaperones (GroEL/ES in G. moniliformis and P. marneffei; GroEL/ES and trigger factor in N. crassa and A. niger CBS 513.88) enhanced the enzyme solubility. Specific activities of strains expressing the former two enzymes increased approximately fourfold upon co-expression of GroEL/ES. The enzyme from G. moniliformis (co-purified with GroEL) preferred benzonitrile as substrate (K(m) of 0.41 mM, V(max) of 9.7 μmol min(-1) mg(-1) protein). The P. marneffei enzyme (unstable in its purified state) exhibited the highest V(max) of 7.3 μmol min(-1) mg(-1) protein in cell-free extract, but also a high K(m) of 15.4 mM, for 4-cyanopyridine. The purified nitrilases from A. niger CBS 513.88 and N. crassa acted preferentially on phenylacetonitrile (K(m) of 3.4 and 2.0 mM, respectively; V(max) of 10.6 and 17.5 μmol min(-1) mg(-1) protein, respectively), and hydrolyzed also (R,S)-mandelonitrile with higher K(m) values. Significant amounts of amides were only formed by the G. moniliformis nitrilase from phenylacetonitrile and 4-cyanopyridine.     

Topogenesis of heterologously expressed fragments of the human Y4 GPCR

Fragments of large membrane proteins have the potential to facilitate structural analysis by NMR, but their folding state remains a concern. Here we determined the quality of folding upon heterologous expression for a series of N- or C-terminally truncated fragments of the human Y4 G-protein coupled receptor, amounting to six different complementation pairs. As the individual fragments lack a specific function that could be used to ascertain proper folding, we instead assessed folding on a basic level by studying their membrane topology and by comparing it to well-established structural models of GPCRs. The topology of the fragments was determined using a reporter assay based on C-terminal green fluorescent protein- or alkaline phosphatase-fusions. N-terminal fusions to Lep or Mistic were used if a periplasmic orientation of the N-terminus of the fragments was expected based on predictions. Fragments fused to Mistic expressed at comparably high levels, whereas Lep fusions were produced to a much lower extent. Though none of the fragments exclusively adopted one orientation, often the correct topology predominated. In addition, systematic analysis of the fragment series suggested that the C-terminal half of the Y4 receptor is more important for adopting the correct topology than the N-terminal part. Using the detergent dodecylphosphocholine, selected fragments were solubilized from the membrane and proved sufficiently stable to allow purification. Finally, as a first step toward reconstituting a functional receptor from two fragments, we observed a physical interaction between complementing fragments pairs upon co-expression.     

Subcellular localization of aphidicolin biosynthetic enzymes heterologously expressed in Aspergillus oryzae

The secondary metabolite aphidicolin has previously been produced by Aspergillus oryzae after the heterologous expression of four biosynthetic enzymes isolated from Phoma betae. In this study, we examined the subcellular localization of aphidicolin biosynthetic enzymes in A. oryzae. Fusion of green fluorescent protein to each enzyme showed that geranylgeranyl diphosphate synthase and terpene cyclase are localized to the cytoplasm and the two monooxygenases (PbP450-1 and PbP450-2) are localized to the endoplasmic reticulum (ER). Protease protection assays revealed that the catalytic domain of both PbP450s was cytoplasmic. Deletion of transmembrane domains from both PbP450s resulted in the loss of ER localization. Particularly, a PbP450-1 mutant lacking the transmembrane domain was localized to dot-like structures, but did not colocalize with any known organelle markers. Aphidicolin biosynthesis was nearly abrogated by deletion of the transmembrane domain from PbP450-1. These results suggest that ER localization of PbP450-1 is important for aphidicolin biosynthesis.     

Isolation and characterization of a heterologously expressed bacterial laccase from the anaerobe Geobacter metallireducens

Applied Microbiology and Biotechnology - Bioinformatics has revealed the presence of putative laccase genes in diverse bacteria, including extremophiles, autotrophs, and, interestingly, anaerobes....     

Patch clamp analysis of a H+ pump heterologously expressed in giant yeast vacuoles

Despite the usefulness of the patch-clamp technique, its application to ion pumps and transporters in biomembranes is limited. We developed a novel method for determining the activity of a proton-pumping pyrophosphatase (H(+)-PPase) made of a single protein. We heterologously highly expressed the enzyme in Saccharomyces cerevisiae, prepared giant vacuoles from the cells, and measured a PPi-dependent electrical current of 18 pA (10.5 fA/ micro m(2)) using the patch-clamp technique in the whole-vacuole recording mode. We determined the inhibitor sensitivity and affinity for substrate (K(m), 4.6 micro M). The enzyme number in a giant vacuole (4.2 x 10(6)) and the molecular activity of the expressed H(+)-PPase (14 s(-1)) were determined. An uncoupling-type H(+)-PPase mutant, of which the 263rd glutamate residue was replaced by aspartate, and of which H(+) pump activity was not detected with the fluorescence quenching method, showed a weak current with a high K(m). The high accuracy, effectiveness and applicability of the method for exogenously expressed ion transporters were also discussed.     

Rapid purification of proline-specific endopeptidase fromFlavobacterium meningosepticum heterologously expressed inEscherichia coli

Two fast and efficient purification methods for the preparation of large amounts of proline-specific endopeptidase (PSE) [EC 3.4.21.26] fromFlavobacterium meningosepticum heterologously expressed inEscherichia coli are described. Overproduction and accumulation of PSE in the periplasmic space ofE. coli means that a single gel chromatography step or ion exchange chromatography step was sufficient to obtain homogenously pure PSE preparations. With these procedures, up to 490 g of purified enzyme per gE. coli cells were obtained. The different purification methods are discussed.The authors are with the Weissheimer Research Laboratory, Department of Biotechnology, Schaarstrasse 1, D-56626 Andernach, Germany     

The 5A structure of heterologously expressed plant aquaporin SoPIP2;1

SoPIP2;1 is one of the major integral proteins in spinach leaf plasma membranes. In the Xenopus oocyte expression system its water channel activity is regulated by phosphorylation at the C terminus and in the first cytosolic loop. To assess its structure, SoPIP2;1 was heterologously expressed in Pichia pastoris as a His-tagged protein and in the non-tagged form. Both forms were reconstituted into 2D crystals in the presence of lipids. Tubular crystals and double-layered crystalline sheets of non-tagged SoPIP2;1 were observed and analyzed by cryo-electron microscopy. Crystalline sheets were highly ordered and diffracted electrons to a resolution of 2.96A. High-resolution projection maps of tilted specimens provided a 3D structure at 5A resolution. Superposition of the SoPIP2;1 potential map with the atomic model of AQP1 demonstrates the generally well conserved overall structure of water channels. Differences concerning the extracellular loop A explain the particular crystal contacts between oppositely oriented membrane sheets of SoPIP2;1 2D crystals, and may have a function in rapid volume changes observed in stomatal guard cells or mesophyll protoplasts. This crystal packing arrangement provides access to the phosphorylated C terminus as well as the loop B phosphorylation site for studies of channel gating.     

Characterization of a functional soluble form of a Brassica napus membrane-anchored endo-1,4-beta-glucanase heterologously expressed in Pichia pastoris

The Brassica napus gene, Cel16, encodes a membrane-anchored endo-1,4-beta-glucanase with a deduced molecular mass of 69 kD. As for other membrane-anchored endo-1,4-beta-glucanases, Cel16 consists of a predicted intracellular, charged N terminus (methionine(1)-lysine(70)), a hydrophobic transmembrane domain (isoleucine(71)-valine(93)), and a periplasmic catalytic core (lysine(94)-proline(621)). Here, we report the functional analysis of Delta(1-90)Cel16, the N terminally truncated Cel16, missing residues 1 through 90 and comprising the catalytic domain of Cel16 expressed recombinantly in the methylotrophic yeast Pichia pastoris as a soluble protein. A two-step purification protocol yielded Delta(1-90)Cel16 in a pure form. The molecular mass of Delta(1-90)Cel16, when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was about 130 kD and about 60 kD after enzymatic removal of N-glycans, fitting the expected molecular mass of 59 kD. Delta(1-90)Cel16 was highly N glycosylated as compared with the native B. napus Cel16 protein. Delta(1-90)Cel16 had a pH optimum of 6.0. The activity of Delta(1-90)Cel16 was inhibited by EDTA and exhibited a strong dependence on calcium. Delta(1-90)Cel16 showed substrate specificity for low substituted carboxymethyl-cellulose and amorphous cellulose. It did not hydrolyze crystalline cellulose, xyloglycan, xylan, (1-->3),(1-->4)-beta-D-glucan, the highly substituted hydroxyethylcellulose, or the oligosaccharides cellotriose, cellotetraose, cellopentaose, or xylopentaose. Size exclusion analysis of Delta(1-90)Cel16-hydrolyzed carboxymethylcellulose showed that Delta(1-90)Cel16 is a true endo-acting glucanase.     

Characterization of heterologously expressed recombinant retinoic acid receptors with natural or synthetic retinoids

The first step in retinoid action is binding to their nuclear receptors. Therefore, characterization of binding characteristics of retinoids is of major importance. Human retinoic acid receptors α (hRARα), hRARβ, and mouse RARγ (mRARγ) were expressed heterologously in Escherichia coli as a recombinant glutathione S-transferase (GST) fusion protein. The expressed fusion proteins were functional and bound specifically to the all-trans-retinoic acid (RA). The dissociation constants (K_d) for RA were 1.4 nM for GST-hRARα, 1.4 nM for GST-hRARβ, and 3.3 nM for GST-mRARγ, respectively. The fusion proteins were further used for competitive displacement assays to determine the displacement constant (DC50) for other selected retinoids. All-trans-RA and 4-oxo-all-trans-RA have high affinity with all three receptors (DC50 = 0.8 ～ 55 nM). The 13-cis RA binds to hRARα with low affinity, but not to other RARs evaluated here. All-trans-N-ethylretinamide, all-trans-retinylacetate, and an ethyl ester of tetrahydronaphthalene derivative had no affinity to any RARs. The hRARα and mRARγ receptors did not bind a naphthalene carboxylic acid derivative of RA, but hRARβ binds this chemical with high affinity. Results indicated that the three recombinant proteins were functional in binding various RA congeners. The affinity and binding data of these retinoids were compared to their observed teratogenic activity.     

pH Dependency and desensitization kinetics of heterologously expressed combinations of acid-sensing ion channel subunits

The exact subunit combinations of functional native acid-sensing ion channels (ASICs) have not been established yet, but both homomeric and heteromeric channels are likely to exist. To determine the ability of different subunits to assemble into heteromeric channels, a number of ASIC1a-, ASIC1b-, ASIC2a-, ASIC2b-, and ASIC3-containing homo- and heteromeric channels were studied by whole-cell patch clamp recordings with respect to pH sensitivity, desensitization kinetics, and level of sustained current normalized to peak current. Analyzing and comparing data for these three features demonstrated unique heteromeric channels in a number of co-expression experiments. Formation of heteromeric ASIC1a+2a and ASIC1b+2a channels was foremost supported by the desensitization characteristics that were independent of proton concentration, a feature none of the respective homomeric channels has. Several lines of evidence supported formation of ASIC1a+3, ASIC1b+3, and ASIC2a+3 heteromeric channels. The most compelling was the desensitization characteristics, which, besides being proton-independent, were faster than those of any of the respective homomeric channels. ASIC2b, which homomerically expressed is not activated by protons per se, did not appear to form unique heteromeric combinations with other subunits and in fact appeared to suppress the function of ASIC1b. Co-expression of three subunits such as ASIC1a+2a+3 and ASIC1b+2a+3 resulted in data that could best be explained by coexistence of multiple channel populations within the same cell. This observation seems to be in good agreement with the fact that ASIC-expressing sensory neurons display a variety of acid-evoked currents.     

Structural characterisation of neuronal voltage-sensitive K+ channels heterologously expressed in Pichia pastoris

Neuronal voltage-dependent K(+) channels of the delayed rectifier type consist of multiple Kv alpha subunit variants, which assemble as hetero- or homotetramers, together with four Kv beta auxiliary subunits. Direct structural information on these proteins has not been forthcoming due to the difficulty in isolating the native K(+) channels. We have overexpressed the subunit genes in the yeast Pichia pastoris. The Kv1.2 subunit expressed alone is shown to fold into a native conformation as determined by high-affinity binding of 125I-labelled alpha-dendrotoxin, while co-expressed Kv1.2 and Kv beta 2 subunits co-assembled to form native-like oligomers. Sites of post-translational modifications causing apparent heterogeneity on SDS-PAGE were identified by site-directed mutagenesis. Engineering to include affinity tags and scale-up of production by fermentation allowed routine purification of milligram quantities of homo- and heteroligomeric channels. Single-particle electron microscopy of the purified channels was used to generate a 3D volume to 2.1 nm resolution. Protein domains were assigned by fitting crystal structures of related bacterial proteins. Addition of exogenous lipid followed by detergent dialysis produced well-ordered 2D crystals that exhibited mostly p12(1) symmetry. Projection maps of negatively stained crystals show the constituent molecules to be 4-fold symmetric, as expected for the octameric K(+) channel complex.     

Activation properties of heterologously expressed mammalian TRPV2: evidence for species dependence

TRPV2 has been proposed as a potential pain target, in part due to its relatedness to the nociceptor TRPV1 and to its reported activation by noxious high temperatures (>52 degrees C). However, TRPV2 responses to heat as well as to the nonselective agonist 2-aminoethoxydiphenyl borate (2-APB) have not been universally reproduced in other laboratories, leading to debate about the activation properties of this channel. Here, we report the expression of rat, mouse, and human TRPV2 in HEK293 cells and the differential properties of their responses to heat and 2-APB. Expression of mouse or rat TRPV2 in HEK293 cells resulted in robust channel activation when induced by either temperature (>53 degrees C) or 2-APB. By contrast, expression of human TRPV2 did not lead to detectable activation by either of these stimuli. Human TRPV2 protein was expressed at levels comparable with those of rat TRPV2, exhibited similar surface localization and responded to a novelly identified TRPV2 agonist, Delta(9)-tetrahydrocannabinol, indicating that human TRPV2 is functionally expressed on the cell surface. Studies using deletion mutants and chimeras between rat and human TRPV2 indicated that both amino- and carboxyl-cytoplasmic termini of rat TRPV2 are important for responses to heat and 2-APB but can be supplied in trans to form an active channel. The present study not only confirms and extends previous reports demonstrating that rat and mouse TRPV2 respond to 2-APB and noxious heat but also indicates that further investigation will be required to elucidate TRPV2 activation and regulatory mechanisms.     

Direct solubilization of heterologously expressed membrane proteins by incorporation into nanoscale lipid bilayers

One of the biggest challenges in the field of proteomics is obtaining functional membrane proteins solubilized and dispersed into a physiologically relevant environment that maintains the spectrum of in vivo activities. Here we describe a system composed of nanoscale self-assembled particles, termed Nanodiscs, which contain a single phospholipid bilayer stabilized by an encircling membrane scaffold protein (MSP). Using microsomal membranes of baculovirus-infected Spodoptera frugiperda (Sf9) insect cells overexpressing an N-terminally anchored cytochrome P450 monoxygenase (P450), we demonstrate that target membrane proteins can be directly solubilized and incorporated into distinct populations of Nanodiscs, which can be separated by size chromatography. We show that formation of these Nanodiscs from insect cell membranes allows for the compartmentalization into soluble nanostructures that provide a natural membrane bilayer that avoids the aggregation of membrane proteins often encountered in other reconstitution procedures. Lipid composition analysis and substrate binding analysis of size-fractionated Nanodiscs arrayed in microtiter plates further demonstrates that the Nanodisc system effectively disperses the overexpressed membrane protein into monodispersed bilayers containing biochemically defined lipid components and the target protein in its native from suitable for sensitive high-throughput substrate binding analysis.     

Isotopic labeling of mammalian G protein-coupled receptors heterologously expressed in Caenorhabditis elegans

High-resolution structural determination and dynamic characterization of membrane proteins by nuclear magnetic resonance (NMR) require their isotopic labeling. Although a number of labeled eukaryotic membrane proteins have been successfully expressed in bacteria, they lack post-translational modifications and usually need to be refolded from inclusion bodies. This shortcoming of bacterial expression systems is particularly detrimental for the functional expression of G protein-coupled receptors (GPCRs), the largest family of drug targets, due to their inherent instability. In this work, we show that proteins expressed by a eukaryotic organism can be isotopically labeled and produced with a quality and quantity suitable for NMR characterization. Using our previously described expression system in Caenorhabditis elegans, we showed the feasibility of labeling proteins produced by these worms with ¹⁵N,¹³C by providing them with isotopically labeled bacteria. ²H labeling also was achieved by growing C. elegans in the presence of 70% heavy water. Bovine rhodopsin, simultaneously expressed in muscular and neuronal worm tissues, was employed as the “test” GPCR to demonstrate the viability of this approach. Although the worms’ cell cycle was slightly affected by the presence of heavy isotopes, the final protein yield and quality was appropriate for NMR structural characterization.